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Increased Excretion of Modified Adenine Nucleosides by Children with Adenosine Dearninase Deficiency

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Increased Excretion of Modified Adenine Nucleosides by Children with Adenosine Dearninase Deficiency Pediatr. Res. 16: 362-369 (1982) Increased Excretion of Modified Adenine Nucleosides by Children with Adenosine Dearninase Deficiency ROCHELLE HIRSCHHORN,'"~ HOWARD RATECH, ARYE RUBINSTEIN, PHOTINI PAPAGEORGIOU, HERNANT KESARWALA, ERWIN GELFAND, AND VIVIEN ROEGNER-MANISCALCO Departments of Medicine and Pathology, New York University School of Medicine, New York, New York [R.H., H.R., and V.R.-M.];Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York [A.R.]; Department of Pediatrics, Rutgers University Medical ~chool,Piscataway, New Jersey [P.P., and H.K.]; and Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada [E. G.] Summary tially in, and prevents proliferation of, irnmunocompetent cells, primarily of the T cell class (2, 5, 6, 23, 38, 49, 54). There is also We have identified seven adenine nucleosides in urines of un- in vivo and/or in vitro evidence for alternative mechanisms of treated adenosine deaminase (ADA) deficient patients, four of toxicity, which would operate via depletion of pyrimidine pools, which (adenosine, 2'-deoxyadenosine, 1-methyladenosine and N6- depletion of phosphoribosyl pyrophosphate and increases in cyclic methyladenosine) have been previously identified in urines of AMP or S-adenosyl homocysteine (16, 21, 24, 40, 46, 55). All of normals and/or ADA deficient patients. We confirm that ADA these mechanisms are dependent on accumulation of the substrates deficient patients excrete markedly increased amounts of 2'-deox- of ADA, adenosine and 2'-deoxyadenosine. yadenosine (582 k 363 versus normal of < 0.1 nmoles/mg creati- In addition to adenosine and 2'-deoxyadenosine, several other nine) and increased amounts of adenosine (29.4 & 5.7 versus modified adenine nucleosides occur naturally (17, 19) and are normal of 4.12 & 1.0 nmoles/mg creatinine). substrates for ADA (1, 18, 43, 56). Such naturally occurring We have found three other modified adenine nucleosides previ- modified adenine nucleosides might be expected to also accumu- ously undetected in human urine. These three compounds are 2'- late in ADA deficient patients and possibly contribute to toxicity. 0-methyladenosine, N6, 2'-O-dimethyladenosine and an as yet incompletely characterized modified adenine nucleoside, R-aden- We have, therefore, sought to determine if these modified adenine nucleosides are uniquely present, or present in increased amounts, osine. Only ADA deficient patients excrete detectable amounts of in urine of ADA deficient patients. We have identified seven 2'-0-methyladenosine (2.1 1.1 versus normal of < 0.1 nmoles/ + compounds in urines of ADA deficients, four of which (adenosine, mg creatinine), whereas both normals and ADA deficient children excrete N6, 2'-Odimethyladenosine and R-adenosine. However, 2'-deoxyadenosine, 1-methyladenosine and N"methy1adenosine) have been previously identified in urines of normals and/or ADA ADA deficient patients do excrete increased amounts of R-aden- deficient patients (4,9,27,28,32,36,37,50). We have additionally osine (5.5 1.0 versus normal of 1.4 k 0.4 nmoles/mg creatinine). + found three other substrates of ADA not ~reviouslvdetected in urine. These three compounds newly identified in urine are 2'-0- Speculation methyladenosine, N6,2'-0-dimethyladenosine and an as yet in- Accumulation in ADA deficient patients of two newly detected completely characterized modified adenine nucleoside, R-adeno- modified adenine nucleosides (2'-0-methyladenosine and R-aden- sine. Only ADA deficient patients excrete detectable amounts of osine) or their metabolites could play a role in explaining the 2'-0-methyladenosine, whereas both normal and ADA deficient profound abnormalities of B cell function seen in ADA deficiency children excrete N"2'-0-dimethyladenosine and the as yet uni- but not in purine nucleoside phosphorylase deficiency. This differ- dentified ADA substrate R-adenosine. However, ADA deficient ential involvement of B cell function is not easily explained by patients excrete increased amounts of R-adenosine. accumulation of deoxytrinucleotides, which occurs in both disor- ders. MATERIALS AND METHODS Materials. Nucleosides were obtained from Sigma Co. (St. Inherited deficiency of the purine salvage enzyme, adenosine Louis, MO) or P&L (Milwaukee, WI). 2'-0-Methylinosine was deaminase (ADA) results in a fatal infantile syndrome of severe generated by incubation of 2'-0-methyladenosine with calf intes- combined immunodeficiency (ADA-SCID) (10, 15, 22, 25, 30). tinal ADA, type I from Sigma, and purified by high pressure Children with ADA deficiency have a profound defect in both liquid chromatography. 5'-Methylthioadenosine was generated by cellular and humoral immunity, although in 10-15% of cases, the acid hydrolysis of ,S-adenosyl methionine (45). 5'-deoxyadenosine humoral defect may initially be less severe (22). Affected children and N~threonino~arbon~ladenosine were the generous gifts of accumulate and/or excrete markedly increasd amounts of the respectively Dr. G. Elion and Dr. G.B. Chheda. ADA substrates, adenosine and 2'-deoxyadenosine, and the phos- Anion exchange chromatography. Urines (stored frozen at phorylated metabolite, deoxy ATP (dATP) (3, 5, 6, 9, 26-28, 32, -70 "C with 0.01% sodium azide) were chromatographed on an 37, 41, 46, 50). anion exchange column (Biorad AG I-X2, Richmond, CA) essen- Several pathophysiologic mechanisms have been proposed tially as described by Kuttesch, et al. (32) except that four fractions whereby accumulation of ADA substrates and their metabolites were collected. Fraction 1, 1-6 ml; fraction 2, 7-3 1 ml; fraction 3, would result in immunodeficiency (5, 12, 15, 16, 21, 24, 40, 52). 32-52 ml and fraction 4, 53-73 ml (eluting after the addition of The largest body of evidence supports the hypothesis that ~ATP, acid). Adenosine and 2'-deoxyadenosine eluted in fraction 2 and an inhibitor of ribonucleotide reductase, accumulates preferen- adenine in fraction 4, as previously described (32, 36). The elution ADENOSINE DEAMINASE DEFICIENCY 363 profiles of several authentic compounds, including modified ade- Peak shifi and acid hydrolysis. Samples and authentic com- nine and guanine nucleosides and bases are shown in Figure I. pounds were incubated with and without calf intestinal ADA High pressure liquid chromarography (HPLC).Urines (fraction- (Sigma Type 1, St. Lfiuis, MO) (20,42) at 0.08, 1.5 and 15 IU/ml ated and unfractionated) were analyzed by HPLC on C 18 pBon- final concentration for 1-2 h at 37OC and then analyzed by HPLC dapak columns (Waters Co., Milford, MA) by methods previously (Fig. 2 and Table 1). For hydrolysis of nucleosides to bases, peaks described (42) except that the gradient went from 0 to 40% of interest were collected by HPLC, concentrated, reconstituted methanol in 60 min. For further identification, peaks of interest with H20 and an aliquot boiled for 30 min in 0.45 N perchloric were collected, dried under nitrogen, reconstituted with water, and acid and neutralized with KOH. The authentic compounds aden- reanalyzed following either treatment with ADA, acid hydrolysis osine, 2'-deoxyadenosine, 2'-0-methyladenosine, N6-methylad- or chromatography on Afigel601 (Biorad, Richmond, CA). UV enosine, I-methyladenosine, N6,N6,-dimethyladenosineand N6- ratios of compounds were determined by simultaneous monitoring isopentenyladenosine were hydrolyzed under the same conditions at the two wavelengths specified. Retention times of relevant and the reaction products analyzed by HPLC. Nz-methylguano- nucleosides and bases are listed in Table 1. sine eluted in the last of the anion exchange fractions (Fig. I) and 0-0 INOSINE r-• N6UETHYL ADENOSINE ( 0-0 OEOXYINOSINE &-d NsH( DIuETnYL ADENOSINE ' W GUANOSINE )-8 2'0 METHYL ADENOSINE r-r GUANINE 0-0 7 METnlL GUlNOSlNE , B--B N~UETHIL GUINOSlNE I - NIM DIMETHYL GUANOS~NEI 4 8 12 16 20 24 28 JL56 60 64 68 72 74 ELUTION VOLUME (ml) Fig. I. Elution of methylated nucleosides from dowex AG I X2. Authentic compounds were diluted and chromatographed on Dowex AG I X2 columns as described in "Materials and Methods". All the modified adenine nucleosides tested (as well as adenosine) eluted in the sewnd fraction except for I-methyladenosine which eluted in the first and sewnd fractions. Isopentenyl adenosine could not be recovered by this method (not shown). Table 1. Relative retention times of mod$ed adenine nucleosides and the products generared by deamination or acid hydrolysis Retention time Retention time2 Retention time2 Retention time Deamination Retention time Retention time Compound Adenosine product Inosine Hydrolysis product Adenin I-Methyladenosine' Adenosine' inosine adenine 2'-Deoxyadenosine' 2'-deoxyinosine adenine N" threonino~arbon~ladeno- NT' NT sine 5'-Deoxyadenosine 5'-deoxyinosine adenine R-adenosine' R-inosine R-adenine 2'-0-Methyladenosine' 2'-0-methylinosine adenine N6-Methyladenosine' inosine N6-methyladenine 3'-0-Methyladenosine 3'-0-methylinosine adenine ~",2'-0-Dimethyladenosine' 2'-0-methylinosine adenine N"N"-Dimethyladenosine inosine N"N6,-dimethyladenine lsopentenyladenosine inosine "isopenteny ladenineW3 5'-Methylthioadenosine NT NT Adenosine N'-oxide NT NT 2-Methyladenosine NT 2-methyladenine - -- ' Compounds isolated from urine as well as authentic compounds, treated with ADA and also acid hydrolyzed. Retention times of urinary compounds were identical to those of the authentic wmpounds, as determined by coelution. Inosine and adenine have retention time relative to adenosine (1.00) of 0.58 and 0.70. All closely eluting
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